Conventional optical microscopy provides high resolution (˜200 nm) images and has a huge range of applications, from inspection of electronic devices to cell biology. In many cases, it is desirable to obtain so-called ‘optically sectioned’ images, i.e. an image of only an axially thin slice through the sample. The advantages of optically sectioned imaging include reduction of out-of-focus blur, a potential increase in resolution, a reduction in light scattered from out-of-focus planes, and an ability to produce high resolution 3D images. The conventional method for obtaining high quality optically sectioned images is confocal microscopy.
Confocal microscopy involves scanning a point of illumination and detecting the reflected or fluorescent light back to a confocal point detector. This results in high quality confocal imaging, but it is necessary to scan the point source and detection region over the sample in two or three dimensions, depending on whether a 2D or 3D image is required. Such scanning can limit the data acquisition rate or, if rapid scanning is employed, will increase the peak power at the sample, which can lead to increased photodamage and phototoxicity of biological samples.
The scanning speed in confocal microscopy can be increased through the use of multiple excitation and detection spots, e.g. in a Nipkow disk microscope. However, the closer adjacent spots are placed, the greater the chance of cross-talk between neighbouring confocal pinholes, which produces a concomitant increase in the size of side lobes or pedestal on the axial point spread function.
A number of alternative methods to confocal microscopy have been proposed, generally termed ‘structured illumination’ techniques. However, these all require the acquisition of multiple images using a CCD camera followed by image processing to calculate the sectioned image. Performing calculations on weak (noisy) fluorescence images leads to a compounding of the noise in the final image. All confocal and structured illumination techniques require that the whole sample be illuminated along its axial extent, even though only a single lateral plane in the sample is being imaged, and this leads to unnecessary photobleaching and phototoxic effects.
A recently developed technique for obtaining optically sectioned images is that of Selective Plane Illumination Microscopy (SPIM) [1,2], which followed early work by Voie et al. [3] and Fuchs et al. [4]. The SPIM technique [5] uses two objective lenses, separated by an angle of 90° relative to one another and used to view the same sample. One lens is used to illuminate only a thin ‘sheet’ within the sample and the second lens is used to produce a diffraction limited image of this sheet. The optical configuration for SPIM is illustrated in FIG. 1. The region in the sample where fluorescence is excited is perfectly imaged by the detection optics onto the detection image plane. It should be noted that the image is stretched axially due to the greater (M2) axial magnification of the detection optical system. SPIM has been used to obtain images of small organisms and embryos and can be used to image both reflected or scattered light and fluorescence [5].
The drawback of SPIM is that two objective lenses are required and this gives rise to the two main disadvantages of this technique. First, it is mechanically difficult to arrange for the two objectives to be placed close enough to one another so that a high numerical aperture lens can be used to collect the light while still being able to produce a thin sheet of illumination. This can restrict the numerical aperture and hence resolution of the imaging system. Second, the need to illuminate the sample with a lens that is in the plane of the sample being imaged means that conventional sample preparation techniques, e.g. glass microscope slides, cannot be used, and a special sample holder needs to be used instead.
Recent work by Tokunaga et al. [6] and Konopka et al. [7] has shown that it is possible to illuminate a thin sheet of a sample using the same objective that is used to collect the fluorescence. This is illustrated in FIG. 2. This imaging system was termed Highly Inclined and Laminated Optical sheet (HILO) microscopy and variable angle epi-fluorescence microscopy. A 3D image of the specimen can then be produced by scanning the sheet illumination or specimen in one direction. This system is nearly equivalent to a SPIM system, but with two significant differences; the illumination and detection beams are not at 90° (as is usual for SPIM) and the sheet of illumination does not align in the focal plane of the imaging system used to collect the reflected/scattered light or fluorescence. This is shown in the image plane of FIG. 2, where the image of the sample fluorescence (shown as a stripe) lies at significant angle to the image plane (dashed line). The detector cannot simply be tilted with respect to the optical axis due to unwanted spherical aberrations that would arise. This aberration will be most severe for parts of the image of the sample image that are furthest from the image plane.
There is therefore a desire to be able to use a technique similar to SPIM, but using a single objective lens at the sample, and with the illumination and detection beams at 90° at the sample, whilst avoiding (or at least minimising) the aberration affects.
Further background art is provided in WO 2008/078083, which discloses a focusing apparatus for use with an optical system. The focusing apparatus includes a focus adjusting means, which enables the position of a selected axial focal plane to be adjusted within the sample.